Total concentration of homocysteine (Hcy) in body fluid, such as plasma or serum, has been known as an important marker for several specific diseases. Since a critical role of Hcy in human body was introduced in the late 1960s, many clinical studies have demonstrated that Hcy is a prognostic maker of cardiovascular disease and pathogenesis of atherosclerosis. Although the pathogenesis mechanism of these diseases by Hcy has not been fully understood, efforts for clearly establishing the role of Hcy in such diseases are ongoing in clinical and basic medical fields. Regarding cardiovascular disease, it is reported that elevated Hcy level triggers increased oxidant stress in the vasculature (Weiss, N. CurrDrug Metab 2005, 6, 27-36; Papatheodorou, L.; Weiss, N. Antioxid Redox Signal 2007, 9, 1941-1958). The elevated Hcy level known as hyperhomocysteinemia is also associated with Alzheimer's disease (Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D'Agostino, R. B.; Wilson, P. W.; Wolf, P. A. N Engl J Med 2002, 346, 476-483; Nilsson, K.; Gustafson, L.; Hultberg, B. Clin Chem Lab Med 2008, 46, 1556-1561; Van Dam, F.; Van Gool, W. A. Arch Gerontol Geriatr 2009, 48, 425-430), Parkinson's disease (Bialecka, M.; Robowski, P.; Honczarenko, K.; Roszmann, A.; Slawek, J. Neurol Neurochir Pol 2009, 43, 272-285), neural tube defects (Molloy, A. M.; Brody, L. C.; Mills, J. L.; Scott, J. M.; Kirke, P. N. Birth Defects Res A Clin Mol Teratol 2009, 85, 285-294), pregnancy complications (Wheeler, S. Proc Nutr Soc 2008, 67, 437-450), and osteoporosis (van Meurs, J. B.; Dhonukshe-Rutten, R. A.; Pluijm, S. M.; van der Klift, M.; de Jonge, R.; Lindemans, J.; de Groot, L. C.; Hofman, A.; Witteman, J. C.; vanLeeuwen, J. P.; Breteler, M. M.; Lips, P.; Pols, H. A.; Uitterlinden, A. G. N Engl J Med 2004, 350, 2033-2041). In fact, the balance between Hcy production and utilization can be disturbed by the clinical states characterized by genetic disorders. Thus, a simple and accurate quantitative analysis method for Hcy is needed to diagnose the above-mentioned diseases and newborn defects in metabolism.
Nowadays, some methods for quantifying Hcy involve traditional analytical techniques such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and immunoassay. These methods have been widely used for Hcy quantification in the clinical monitoring of biological fluids, however, they have the disadvantages of complication and inefficiency of labeling, poor stability, and relatively low intensities of dyes. In addition, they require expensive and highly specialized analysis equipment and trained operators, and thus they are often costly, and time-consuming to perform. Accordingly, there is a need to develop a simple, cheap, and highly accurate and sensitive Hcy quantification method in order to effectively detect and quantify Hcy, a disease marker.
For the development, we performed studies on self-luminescent/fluorescent Hcy/methionine auxotroph microorganism having optimal growth characteristics under assay environments, and Hcy/methionine quantitative analysis methods based on the microorganism, and finally developed a microorganism that grows in proportion to the amount of Hcy/methionine in a sample and exhibits a luminescent or fluorescent signal by the proportional amount of the growth.